The tripeptide glutathione (hereinafter abbreviated as GSH) is the most prevalent intracellular non-protein sulfhydryl found in biological systems [Meister and Anderson, Ann. Rev. Biochem. 52:711-60 (1983)]. The importance of GSH can be inferred from its many functions and its occurrence in a wide variety of organisms [Larson et al., Eds., Functions of Glutathione, Raven Press, New York (1983)]. Significant GSH levels have been detected in both unicellular and multicellular eukaryotes, as well as in plants and prokaryotic cells. In all of these different cell types, GSH functions either directly or indirectly in a number of enzymatic and metaboli processes. To date, known GSH functions include reductive processes essential for protein synthesis and degradation, formation of precursors necessary for DNA synthesis, various enzymatic and regulating activities, cellular transport systems, and protection of cells from oxidative stress generated by free radicals and reactive oxygen compounds. GSH is a coenzyme for several enzyme systems. GSH is also involved in the detoxification of a number of drugs, e.g., antineoplastic compounds and in the metabolism of some endogenous products, e.g., estrogens, leukotrienes, prostaglandins.
Recent studies have shown that GSH may have a prominent role in a number of other biological processes such as radiosensitivity, cancer therapy, oxygen toxicity, and modulation of immune responses. The study of GSH's functions extends to such diversified fields as toxicology, pharmacology, endocrinology, microbiology and agriculture [Meister and Anderson, Ann. Rev. Biochem. 52:711-60 (1983); Larson et al., Eds., Functions of Glutathione. Raven Press, New York (1983)].
GSH (L-.gamma.-glutamyl-L-cysteinyglycine) is synthesized intracellularly in a series of enzymatic reactions of the .gamma.-glutamyl cycle. In addition to the synthesis of GSH, the .gamma.-glutamyl cycle is responsible for the extracellular transport of GSH and the GSH dependent transport of amino acids into the cell via .gamma.-glutamyl transpeptidase. The combination of these reactions account for the cellular turnover of GSH [Meister, Science 220:472-77 (1983)].
GSH plays a major role in cellular oxidation-reduction reactions. The majority of intracellular GSH is found in the reduced state in cells under normal steady state conditions. Reduced GSH is reversibly oxidized either enzymatically by GSH-peroxidase or non-enzymatically to form the disulfide (hereinafter abbreviated as GSSG). A high ratio of GSH to GSSG is maintained in the cell; however, in cells undergoing oxidative stress, this ratio is altered. The high ratio is restored intracellularly by NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) dependent GSSG reductase. There is no known extracellular mechanism for the reduction of GSSG; therefore, GSH must be supplied continuously by the cell to the extracellular environments [Giffith and Meister, Proc. Natl. Acad. Sci. (U.S.A.) 76:5606-10 (1979)]. The continuous transport of GSH into extracellular compartments suggests that it may have a role in the protection of cell membranes.
The sulfhydryl moiety of GSH assumes primary importance in protecting cells from oxidative insults. Cells are continuously exposed to a variety of endogenous and exogenous oxidants. Peroxides and free oxygen radicals are the most common since these are generated during normal metabolic processes as well as during exposure to ionizing radiation and/or increased oxygen tension. It is known that cellular GSH is the key to the oxygen effect in radiation damage [Biaglow et al., Radiat. Res. 95:437-55 (1983); Morse and Dahl, Nature 271:660-61 (1978); Stankova et al., J. Reticuloendothel. Soc. 21:97-102 (1977); Kowasaki, Intl. J. Radiat. Biol. 32:577-81 (1977)]. Oxidative stress is also imposed through the administration of drugs such as BCNU, adriamycin, and daunomycin and exposure to thiol alkylators such as organometals and alkyl halides [Arrick and Nathan, Cancer Res. 44:4224-32 (1984)]. Although other cellular mechanisms also exist for the removal of reactive oxygen intermediates, the participation of GSH, either directly or indirectly, in the oxidation-reduction cycle is of major importance in the maintenance of cellular function and structure.
GSH undergoes nucleophilic attack by a large variety of endogenous and exogenous compounds to form GSH conjugates. In this type of reaction, the reduced sulfhydryl group of GSH reacts with an electrophile to form a thioether. This reaction may occur spontaneously or may be catalyzed by GSH-S-transferases. GSH conjugates are readily transported across the cell membrane and converted to mercapturic acids in a series of reactions involving .gamma.-glutamyl transpeptidase. Detoxification of foreign compounds (e.g., halogenated hydrocarbons, metabolites of ethanol and aromatic hydrocarbons) through this mechanism results in the formation of N-acetyl-S-substituted cysteines (mercapturic acids) which are ultimately excreted in the urine and feces.
Conjugates of GSH with endogenous compounds also occur under normal conditions and play an important role in the subsequent metabolism and activity of these compounds. For example, conversion to the .DELTA..sup.5 -3-ketosteroids to the corresponding .alpha., .beta.-unsaturated at .DELTA..sup.4 -3-ketosteroids is catalyzed by liver proteins that appear to be identical with GSH-S-transferases [Meister and Anderson, ibid].
GSH also functions in several aspects of hormone, prostaglandin and leukotriene metabolism. For example, the reaction of the epoxide, leukotriene A, with GSH results in the formation of leukotriene C whose activity is different than the parent compound.
GSH may play numerous roles in cell transformation and tumor growth [Novi, Science 212:541-42 (1981); Zucker et al , FEBS Letters 155:107-11 (1983)]. Although ionizing radiation can inhibit proliferation of normal and neoplastic cells, low doses of ionizing radiation (&lt;1000R) can produce the opposite effect, that is, induction of transformation. These inverse effects point out the need to better understand the mechanisms involved. Since antioxidants inhibit both effects, the ionizing radiation probably modulates thiol-dependent phenomena [(Arrick et al., J. Biol. Chem. 257:1231-37 (1984)]. The intracellular GSH levels are good correlates of radiosensitivity and may provide information on cell regulation of growth [Kennedy et al., Carcinogenesis 5:1213-18 (1984)]. Normal murine lymphocytes have 334.+-.25 amole GSH/cell [Noelle and Lawrence, Biochem. J. 198:571 (1981)] and variants of a murine leukemic cell line (L5178Y) that are radioresistant (Do=84R) and radiosensitive (DO=35R) have 2540 and 870 amole GSH/cell, respectively [Alexander et al., In: Cellular Radiation Biology, Williams and Wilkins, Baltimore, p. 241, (1965)].
The involvement of cellular thiols in the regulatory mechanisms necessary for cell proliferation are well established. It has been suggested that thiol related reactions are involved in cancers; glyoxal derivatives (keto-aldehydes) inhibit cell proliferation and tumors lack keto-aldehydes [Szent-Gyorgyi, Science 155:539-41 (1967)]. Since keto-aldehydes are eliminated enzymatically by glyoxalases which utilize GSH, cellular thiols play a prominent role in regulating the concentration of these cancerostatic substances. Of course, the suggestion that glyoxal derivatives may be regulators of cell division and that regulation of their intracellular concentrations may be involved in cancer development is only one of many theories which have been suggested as an explanation for cancers. However, other theories also directly or indirectly have implicated cellular thiols in regulation or lack of regulation of cell growth [Lieberman and Gordon-Smith, Brit. J. Haemat. 44:425-30 (1980)]. Numerous studies have reported that blood GSH levels increase when animals or humans develop tumors, but the basis for this increase or the source of the GSH has not been delineated [Mills et al., J. Nutrition 3:1586-92 (1981)].
GSH may be involved in the development of autoimmune diseases. Numerous drugs used in the treatment of rheumatoid arthritis are thiol-reactive (e.g., d-penicillamine, levamisole, gold salts, and chloroquine) which suggest that thiols may be involved in the development or modulation of autoimmune diseases [Munthe et al., J. Rheumatol. (Suppl.) 7:14-19 (1978); Huck et al., J. Rheum. 11:605-09 (1984); Guber et al., Biochem. Pharm. 16:115-23 (1967)]. It has been reported that clinical improvement of rheumatoid arthritis by treatment with d-pencillamine can be predicted if there is an increase in intracellular GSH. Additionally, GSH levels are known to vary in patients with diabetes and some forms of diabetes are considered autoimmune diseases. GSH also is known to decrease with age and the incidence of autoimmunity increases with age.